Anderson K S, Sikorski J A, Johnson K A
Monsanto Agricultural Company, A Unit of Monsanto Company, St. Louis, Missouri 63167.
Biochemistry. 1988 Sep 20;27(19):7395-406. doi: 10.1021/bi00419a034.
Direct evidence for an enzyme-bound intermediate in the EPSP synthase reaction pathway has been obtained by rapid chemical quench-flow studies. The transient-state kinetic analysis has led to the following complete scheme: (formula; see text) Values for all 12 rate constants were obtained. Substrate trapping experiments in the forward and reverse reactions established the kinetically preferred order of binding and release of substrates and products and showed that shikimate 3-phosphate (S3P) and 5-enolpyruvoylshikimate 3-phosphate (EPSP) dissociate at rates greater than turnover in each direction. Pre-steady-state bursts of product formation were observed in the reaction in each direction indicating a rate-limiting step following catalysis. Single turnover experiments with enzyme in excess over substrate demonstrated the formation of a transient intermediate in both the forward and reverse reactions. In these experiments, the enzymatic reaction was observed by employing a radiolabel in the enol moiety of either phosphoenol pyruvate (PEP) or EPSP. The separation and quantitation of reaction products were accomplished by HPLC monitoring radioactivity. The intermediate was observed as the transient production of radiolabeled pyruvate, formed due to the breakdown of the intermediate in the acid quench used to stop the reaction. The intermediate was observed within 5-10 ms after the substrates were mixed with enzyme and decayed in a reaction paralleling the formation of product in each direction. Thus, the kinetics demonstrate directly the kinetic competence of the presumed intermediate. No pyruvate was formed, on a time scale which is relevant to catalysis, after incubation of the enzyme with dideoxy-S3P and PEP or with EPSP in the absence of phosphate; and so, the intermediate does not accumulate under these conditions. The intermediate broke down to form PEP and EPSP in addition to pyruvate when the reaction was quenched with base rather than acid; therefore, the intermediate must contain the elements of each product. Other experiments were designed to measure directly the phosphate binding rate and further constrain the PEP binding rate. The overall solution equilibrium constant in the forward direction was determined to be 180 by quantitation of radiolabeled reactants and products in equilibrium after incubation with a low enzyme concentration. The internal, active site equilibrium constant was obtained by incubation of radiolabeled S3P with excess enzyme and high concentrations of phosphate and PEP to provide the ratio of [EPSP]/[S3P] = 2.3, which is largely a measure of K4.(ABSTRACT TRUNCATED AT 250 WORDS)
通过快速化学淬灭流动研究获得了在EPSP合酶反应途径中酶结合中间体的直接证据。瞬态动力学分析得出了以下完整的反应历程：（公式；见正文）获得了所有12个速率常数的值。正向和反向反应中的底物捕获实验确定了底物和产物结合与释放的动力学优先顺序，并表明磷酸莽草酸（S3P）和5-烯醇丙酮酸莽草酸-3-磷酸（EPSP）在每个方向上的解离速率都大于周转速率。在每个方向的反应中都观察到了预稳态产物形成的突发，这表明催化之后存在一个限速步骤。底物过量时酶的单周转实验证明了正向和反向反应中均形成了瞬态中间体。在这些实验中，通过在磷酸烯醇丙酮酸（PEP）或EPSP的烯醇部分使用放射性标记来观察酶促反应。通过HPLC监测放射性来完成反应产物的分离和定量。观察到中间体是放射性标记丙酮酸的瞬态产生，这是由于用于终止反应的酸淬灭中中间体的分解所致。在底物与酶混合后5-10毫秒内观察到中间体，并在与每个方向上产物形成平行的反应中衰减。因此，动力学直接证明了假定中间体的动力学活性。在与双脱氧-S3P和PEP一起孵育酶后，或者在没有磷酸盐的情况下与EPSP孵育后，在与催化相关的时间尺度上没有形成丙酮酸；因此，在这些条件下中间体不会积累。当用碱而不是酸淬灭反应时，中间体除了分解形成丙酮酸外，还分解形成PEP和EPSP；因此，中间体必定包含每种产物的成分。设计了其他实验来直接测量磷酸盐结合速率，并进一步限制PEP结合速率。通过在低酶浓度下孵育后对平衡状态下放射性标记反应物和产物进行定量，确定正向反应的总体溶液平衡常数为180。通过将放射性标记的S3P与过量的酶以及高浓度的磷酸盐和PEP一起孵育，以提供[EPSP]/[S3P]=2.3的比率来获得内部活性位点平衡常数，这在很大程度上是对K4的测量。（摘要截断于250字）
